The invention relates to farnesyl-protein transferase inhibitors.
Ras proteins transduce extracellular signals to the nucleus and are part of a large superfamily of GTP-binding proteins that are active when bound to GTP and inactive when bound to GDP. After stimulation by receptor activation, Ras protein binds GTP and transmits a proliferative signal to the nucleus. Hydrolysis of GTP to GDP by a GTPase returns Ras to an inactive state. There are three human ras genes, Harvey (H), Kirsten (K) and N-ras, that each encode a 21 kDa Ras protein. Two splice variants of K-ras, K-4A and K-4B, also exist. Approximately 30% of all human cancers harbor ras mutations that typically impair GTP-ase activity, rendering Ras protein locked in a GTP-bound or active state. Kelloff, G. J. et al., (1997), Cancer Epidemiol. Biomarkers Prev., 6(4):267-282.
Ras proteins are initially synthesized as cytoplasmic, soluble proteins. Post-translational modifications serve to attach Ras protein to the plasma membrane. The amino acid sequences of Ras proteins all end in CAAX, where C is cysteine, A is an aliphatic amino acid and X is another amino acid. The first post-translational modification of Ras protein is attachment of a lipophilic 15-carbon farnesyl moiety (farnesyl pyrophosphate, FPP) to the cysteine residue of the CAAX moiety through a farnesyl-protein transferase (FPT) catalyzed reaction. Subsequently, the AAX tripeptide is proteolytically cleaved and the farnesylated cysteine is converted to its methyl ester. Additional lipid modifications of upstream residues further stabilize the association of Ras protein with the plasma membrane. Lemer, E. C. et al., (1997), Anticancer Drug Des., 12:229-238. A related enzyme, geranylgeranyltransferase (GGT) can transfer a 20-carbon geranylgeranyl moiety to the cysteine residue of the CAAX moiety when X is leucine.
It has been shown that inhibition of FPT blocks the anchorage-independent growth of fibroblasts transformed with ras mutants and results in other morphological changes and down-regulation of Ras-protein activated signalling cascades. Omer, Ch. A. et al., (1997), BioFactors, 6:359-366.
A number of farnesylation inhibitors have been developed. One class of inhibitors includes FPP competitive inhibitors that bind to FPT at the FPP binding site. Compounds of this group include synthetic analogs of FPP such as (xcex1-hydroxyfarnesyl)phosphonic acid, amide analogs, hydroxamate analogs, pivolyloxymethyl ester analogs and difluorinated xcex2-ketophosphonic acid, as well as natural products such as actinoplanic acids, chaetomellic acids, manumycin, perillyl alcohol, d-limonene and metabolites, RPR113228 and zaragozic acid. Many of these compounds are selective for GGT rather than FPT and are inactive in whole cells. Perillyl alcohol and d-limonene have chemopreventive activity and reduce tumor size in animals.
A second class of inhibitors includes CAAX tetrapeptides that can serve as substrates and/or competitive inhibitors of FPT. Structure activity analysis has indicated that nonfarnesylated tetrapeptides containing an aromatic amino acid at the A2 position of CA1A2X and a positive charge on the cysteine amino group are the most potent. Tetrapeptides have limited use in vivo since they are inactive in whole cells. A variety of peptidomimetics that are highly selective for FPT and are potent FPT inhibitors have also been synthesized. In most of these compounds, the peptide backbone was modified to increase stability. To increase membrane penetration, the C-terminal carboxylate was masked in certain compounds using an ester prodrug strategy. Many of these compounds inhibit H-Ras processing in cells and also suppress growth of tumors in animals. For a review of peptidomimetics, see Qian, Y. et al., (1997), Biopolymers, 43:25-41.
There are additional FPT inhibitors including barceloneic acid, cylindrol A, fusidienol, patulin, preussomerins and streptonigrins that have been identified from natural products. The mechanism of action of these compounds is unknown. Kelloff, G. J. et al, 1997, supra.
The invention provides novel phosphosesquiterpenes that function as farnesyl-protein transferase inhibitors. The phosphorylated compounds can be used as effective chemotherapeutic agents with fewer side effects than typically follow from use of other chemotherapeutic agents.
In one aspect, the invention features a substantially pure preparation of a phosphosesquiterpene. The phosphosesquiterpene is a phosphorylation product of a sesquiterpene lactone selected from the group consisting of ambrosanolides, psilostachyanolides, cadinanolides, eremanolides, xanthanolides, guaianolides, germacranolides, elamanolides and eudesmanolides.
The phosphosesquiterpene can be a phosphorylation product of the sesquiterpene lactone having formula A. 
In formula A, C1 is further bonded to hydrogen, hydroxy, short chain alkanoyloxy, or C2 or C10 via a double bond or an oxy linkage. C2 is further bonded to at least one of the following substituents independently selected from the group consisting of hydrogen, hydroxy, oxo, short chain alkanoyloxy, and C1 or C3 via a double bond or an oxy linkage. C3 is further bonded to at least one of the following substituents independently selected from the group consisting of hydrogen, oxo, and C2 or C4 via a double bond or an oxy linkage. C4 is further bonded to hydrogen, hydroxy, short chain alkanoyloxy, or C3, C5, or C15 via an oxy linkage or a double bond. C5 is further bonded to hydrogen, hydroxy, short chain alkanoyloxy, or C4 via an oxy linkage or a double bond. C6 is further bonded to hydrogen and C7 is further bonded to hydrogen or C11 via a double bond. C8 is further bonded to at least one of the following substituents independently selected from the group consisting of hydrogen, hydroxy, and short chain alkanoyloxy. C9 is further bonded to at least one of the following substituents independently selected from the group consisting of hydrogen, and C10 via a double bond. C10 is further bonded to hydrogen, hydroxy, short chain alkanoyloxy, C1, or C14 via an oxy linkage, or C1, C9, or C14 via a double bond. C11 is further bonded to hydrogen, hydroxy, hydroxymethyl, or C7 or C13 via a double bond. C13 is further bonded to at least one of the following substituents independently selected from the group consisting of hydrogen, an optionally substituted alkylamino salt, and C11 via a double bond. C14 is further bonded to at least one of the following substituents independently selected from the group consisting of hydrogen and C10 via an oxy linkage or a double bond. C15 is further bonded to at least one of the following substituents independently selected from the group consisting of hydrogen and C4 via an oxy linkage or a double bond.
The phosphosesquiterpene can be a phosphorylation product of the sesquiterpene lactone having formula B. 
In formula B, C1 is further bonded to hydrogen, hydroxy, short chain alkanoyloxy, or C2 or C10 via a double bond or an oxy linkage. C2 is further bonded to at least one of the following substituents independently selected from the group consisting of hydrogen, hydroxy, oxo, short chain alkanoyloxy, and C1 or C3 via a double bond or an oxy linkage. C3 is further bonded to at least one of the following substituents independently selected from the group consisting of hydrogen, oxo, and C2 or C4 via a double bond or an oxy linkage. C4 is further bonded to at least one of the following substituents independently selected from the group consisting of hydrogen, hydroxy, short chain alkanoyloxy, and C3 via an oxy linkage or a double bond. C6 is further bonded to at least one of the following substituents independently selected from the group consisting of hydrogen, hydroxy, and short chain alkanoyloxy. C7 is further bonded to hydrogen or C11 via a double bond and C8 is further bonded to hydrogen. C9 is further bonded to at least one of the following substituents independently selected from the group consisting of hydrogen and C10 via a double bond. C10 is further bonded to hydrogen, hydroxy, short chain alkanoyloxy, C1 or C14 via an oxy linkage and C1, C9, or C14 via a double bond. C11 is further bonded to hydrogen, hydroxy, hydroxymethyl, or C7 or C13 via a double bond. C13 is further bonded to at least one of the following substituents independently selected from the group consisting of hydrogen, an optionally substituted alkylamino salt, and C11 via a double bond. C14 is further bonded to at least one of the following substituents independently selected from the group consisting of hydrogen and C10 via an oxy linkage or a double bond and C15 is further bonded to three hydrogens.
The phosphosesquiterpene can be a phosphorylation product of the sesquiterpene lactone having formula C. 
In formula C, C1 is further bonded to at least one of the following substituents independently selected from the group consisting of hydrogen, hydroxy, oxo, short chain alkyl, short chain alkanoyloxy, C5 via a single bond, C2 via a double bond, and C10 via an oxy linkage or a double bond. C2 is further bonded to at least one of the following substituents independently selected from the group consisting of hydrogen, oxo, hydroxy, short chain alkanoyloxy, and C1 or C3 via a double bond. C3 is further bonded to at least one of the following substituents independently selected from the group consisting of hydrogen, hydroxy, short chain alkanoyloxy, oxo, C4 via an oxy linkage, and C2 or C4 via a double bond. C4 is further bonded to hydrogen, short chain alkanoyloxy, hydroxy, or C3, C5, or C15 via an oxy linkage or a double bond. C5 is further bonded to at least one of the following substituents independently selected from the group consisting of hydrogen, hydroxy, short chain alkanoyloxy, C10 or C1 via a single bond, and C4 via an oxy linkage or a double bond. C6 is further bonded to hydrogen and C7 is further bonded to hydrogen, or C8 or C11 via a double bond. C8 is further bonded to at least one of the following substituents independently selected from the group consisting of hydrogen, hydroxy, short chain alkanoyloxy, and C7 via a double bond. C9 is further bonded to at least one of the following substituents independently selected from the group consisting of hydrogen, short chain alkanoyloxy, and C10 via a double bond. C10 is further bonded to at least one of the following substituents independently selected from the group consisting of hydrogen, C1 or C14 via an oxy linkage, C5 via a single bond, and C1 or C9, C14 via a double bond. C11 is further bonded to hydrogen, hydroxy, hydroxymethyl, or C7 or C13 via a double bond. C13 is further bonded to at least one of the following substituents independently selected from the group consisting of hydrogen, an optionally substituted alkylamino salt, and C11 via a double bond. C14 is further bonded to at least one of the following substituents independently selected from the group consisting of hydrogen and C10 via an oxy linkage or a double bond. C15 is further bonded to at least one of the following substituents independently selected from the group consisting of hydrogen and C4 via an oxy linkage or a double bond.
The phosphosesquiterpene can be a phosphorylation product of the sesquiterpene lactone having formula D. 
In formula D, C1 is further bonded to at least one of the following substituents independently selected from the group consisting of hydrogen, hydroxyl, C10 or C5 via an oxy linkage, and C10 or C2 via a double bond. C2 is further bonded to at least one of the following substituents independently selected from the group consisting of hydrogen, and C1 via a double bond. C3 is further bonded to at least one of the following substituents independently selected from the group consisting of hydrogen, hydroxy, short chain alkanoyloxy, and oxo. C4 is further bonded to hydrogen, hydroxy, short chain alkanoyloxy, or C5 or C15 via an oxy linkage or a double bond. C5 is further bonded to at least one of the following substituents independently selected from the group consisting of hydrogen, short chain alkanoic acid, C1 or C4 via an oxy linkages C10 via a single bond, and C4 or C6 via a double bond. C6 is further bonded to at least one of the following substituents independently selected from the group consisting of hydrogen, hydroxy, short chain alkanoyloxy, and C5 via a double bond. C7 is further bonded to hydrogen, or C11 via a double bond, C8 is further bonded to hydrogen and C9 is further bonded to two hydrogens. C10 is further bonded to hydrogen, C5 via a single bond, or C1 or C14 via an oxy linkage or a double bond. C11 is further bonded to hydrogen, hydroxy, hydroxymethyl, or C7 or C13 via a double bond. C13 is further bonded to at least one of the following substituents independently selected from the group consisting of hydrogen, an optionally substituted alkylamino salt, and C11 via a double bond. C14 is further bonded to at least one of the following substituents independently selected from the group consisting of hydrogen and C10 via an oxy linkage or a double bond. C15 is further bonded to at least one of the following substituents independently selected from the group consisting of hydrogen and C4 via an oxy linkage or a double bond.
The invention also features methods for inhibiting farnesyl-protein transferase. The method includes contacting a cell with an amount of a sesquiterpene lactone effective to inhibit farnesyl-protein transferase activity of the cell. The method can also include monitoring farnesyl-protein transferase activity. The sesquiterpene lactone can be selected from the group consisting of ambrosanolides, psilostachyanolides, cadinanolides, eremanolides, xanthanolides, guaianolides, germacranolides, elamanolides and eudesmanolides. A sesquiterpene lactone having the structure of formula A, B, C or D is particularly useful for inhibiting farnesyl-protein transferase activity.
In another aspect, the invention features a substantially pure preparation of a phosphosesquiterpene. The phosphosesquiterpene can have a skeletal structure of a compound selected from the group consisting of ambrosanolides, psilostachyanolides, cadinanolides, eremanolides, xanthanolides, guaianolides, germacranolides, elamanolides and eudesmanolides. The phosphosesquiterpene can include a gamma-lactone ring or a carboxylic acid substituent.
The phosphosesquiterpene can have formula E. 
C1 of formula E is further bonded to hydrogen, hydroxy, short chain alkanoyloxy, or C2 or C10 via a double bond or an oxy linkage. C2 is further bonded to at least one of the following substituents independently selected from the group consisting of hydrogen, hydroxy, oxo, short chain alkanoyloxy, and C1 or C3 via a double bond or an oxy linkage. C3 is further bonded to at least one of the following substituents independently selected from the group consisting of hydrogen, oxo, and C2 or C4 via a double bond or an oxy linkage. C4 is further bonded to hydrogen, hydroxy, short chain alkanoyloxy, or C3, C5, or C15 via an oxy linkage or a double bond. C5 is further bonded to hydrogen, hydroxy, short chain alkanoyloxy, or C4 via an oxy linkage or a double bond. C6 is further bonded to hydrogen and C7 is further bonded to hydrogen or C11 via a double bond. C8 is further bonded to at least one of the following substituents independently selected from the group consisting of hydrogen, hydroxy, and short chain alkanoyloxy. C9 is further bonded to at least one of the following substituents independently selected from the group consisting of hydrogen, and C10 via a double bond. C10 is further bonded to hydrogen, hydroxy, short chain alkanoyloxy, C1 or C14 via an oxy linkage, or C1, C9, or C14 via a double bond. C11 is further bonded to hydrogen, hydroxy, hydroxymethyl, or C7 or C13 via a double bond. C13 is further bonded to at least one of the following substituents independently selected from the group consisting of hydrogen, an optionally substituted alkylamino salt, and C11 via a double bond. C14 is further bonded to at least one of the following substituents independently selected from the group consisting of hydrogen and C10 via an oxy linkage or a double bond. C15 is further bonded to at least one of the following substituents independently selected from the group consisting of hydrogen and C4 via an oxy linkage or a double bond. M+ is any combination of chemical entities which is able to neutralize any negative charges of the phosphosesquiterpene.
The phosphosesquiterpene can have formula F. 
In formula F, C1 is further bonded to hydrogen, hydroxy, short chain alkanoyloxy, or C2 or C10 via a double bond or an oxy linkage. C2 is further bonded to at least one of the following substituents independently selected from the group consisting of hydrogen, hydroxy, oxo, short chain alkanoyloxy, and C1 or C3 via a double bond or an oxy linkage. C3 is further bonded to at least one of the following substituents independently selected from the group consisting of hydrogen, oxo, and C2 or C4 via a double bond or an oxy linkage. C4 is further bonded to at least one of the following substituents independently selected from the group consisting of hydrogen, hydroxy, short chain alkanoyloxy, and C3 via an oxy linkage or a double bond. C6 is further bonded to at least one of the following substituents independently selected from the group consisting of hydrogen, hydroxy, and short chain alkanoyloxy. C7 is further bonded to hydrogen or C11 via a double bond and C8 is further bonded to hydrogen. C9 is further bonded to at least one of the following substituents independently selected from the group consisting of hydrogen and C10 via a double bond. C10 is further bonded to hydrogen, hydroxy, short chain alkanoyloxy, C1 or C14 via an oxy linkage and C1, C9, or C14 via a double bond. C11 is further bonded to hydrogen, hydroxy, hydroxymethyl, or C7 or C13 via a double bond. C13 is further bonded to at least one of the following substituents independently selected from the group consisting of hydrogen, an optionally substituted alkylamino salt, and C11 via a double bond. C14 is further bonded to at least one of the following substituents independently selected from the group consisting of hydrogen and C10 via an oxy linkage or a double bond. C15 is further bonded to three hydrogens. M+ is any combination of chemical entities which is able to neutralize any negative charges of the phosphosesquiterpene.
The phosphosesquiterpene can have formula G. 
In formula G, C1 is further bonded to at least one of the following substituents independently selected from the group consisting of hydrogen, hydroxy, oxo, short chain alkyl, short chain alkanoyloxy, C5 via a single bond, C2 via a double bond, and C10 via an oxy linkage or a double bond. C2 is further bonded to at least one of the following substituents independently selected from the group consisting of hydrogen, oxo, hydroxy, short chain alkanoyloxy, and C1 or C3 via a double bond. C3 is further bonded to at least one of the following substituents independently selected from the group consisting of hydrogen, hydroxy, short chain alkanoyloxy, oxo, C4 via an oxy linkage, and C2 or C4 via a double bond. C4 is further bonded to hydrogen, short chain alkanoyloxy, hydroxy, or C3, C5, or C15 via an oxy linkage or a double bond. C5 is further bonded to at least one of the following substituents independently selected from the group consisting of hydrogen, hydroxy, short chain alkanoyloxy, C10 or C1 via a single bond, and C4 via an oxy linkage or a double bond. C6 is further bonded to hydrogen and C7 is further bonded to hydrogen, or C8 or C11 via a double bond. C8 is further bonded to at least one of the following substituents independently selected from the group consisting of hydrogen, hydroxy, short chain alkanoyloxy, and C7 via a double bond. C9 is further bonded to at least one of the following substituents independently selected from the group consisting of hydrogen, short chain alkanoyloxy, and C10 via a double bond. C10 is further bonded to at least one of the following substituents independently selected from the group consisting of hydrogen, C1 or C14 via an oxy linkage, C5 via a single bond, and C1 or C9, C14 via a double bond. C11 is further bonded to hydrogen, hydroxy, hydroxymethyl, or C7 or C13 via a double bond. C13 is further bonded to at least one of the following substituents independently selected from the group consisting of hydrogen, an optionally substituted alkylamino salt, and C11 via a double bond. C14 is further bonded to at least one of the following substituents independently selected from the group consisting of hydrogen and C10 via an oxy linkage or a double bond. C15 is further bonded to at least one of the following substituents independently selected from the group consisting of hydrogen and C4 via an oxy linkage or a double bond. M+ is any combination of chemical entities which is able to neutralize any negative charges of the phosphosesquiterpene.
The phosphosesquiterpene can have formula H. 
In formula H, C1 is further bonded to at least one of the following substituents independently selected from the group consisting of hydrogen, hydroxyl, C10 or C5 via an oxy linage, and C10 or C2 via a double bond. C2 is further bonded to at least one of the following substituents independently selected from the group consisting of hydrogen, and C1 via a double bond. C3 is further bonded to at least one of the following substituents independently selected from the group consisting of hydrogen, hydroxy, short chain alkanoyloxy, and oxo. C4 is further bonded to hydrogen, hydroxy, short chain alkanoyloxy, or C5 or C15 via an oxy linkage or a double bond. C5 is further bonded to at least one of the following substituents independently selected from the group consisting of hydrogen, short chain alkanoic acid, C1 or C4 via an oxy linkage, C10 via a single bond, and C4 or C6 via a double bond. C6 is further bonded to at least one of the following substituents independently selected from the group consisting of hydrogen, hydroxy, short chain alkanoyloxy, and C5 via a double bond. C7 is further bonded to hydrogen, or C11 via a double bond and C8 is further bonded to hydrogen. C9 is further bonded to two hydrogens and C10 is further bonded to hydrogen, C5 via a single bond, or C1 or C14 via an oxy linkage or a double bond. C11 is further bonded to hydrogen, hydroxy, hydroxymethyl, or C7 or C13 via a double bond. C13 is further bonded to at least one of the following substituents independently selected from the group consisting of hydrogen, an optionally substituted alkylamino salt, and C11 via a double bond. C14 is further bonded to at least one of the following substituents independently selected from the group consisting of hydrogen and C10 via an oxy linkage or a double bond. C15 is further bonded to at least one of the following substituents independently selected from the group consisting of hydrogen and C4 via an oxy linkage or a double bond. M+ is any combination of chemical entities which is able to neutralize any negative charges of the phosphosesquiterpene.
The invention also relates to a pharmaceutical composition in unit dosage form that includes a phosphosesquiterpene. The composition is suitable for the treatment of a human cancer including breast, colon, rectal, stomach, pancreatic, lung, liver, ovarian, leukemia, lymphoma, pancreatic and esophageal cancer. The composition is particularly useful for the treatment of lung, liver, and ovarian cancer. The phosphosesquiterpene can be phosphorylated dimethylaminoarglabin or a pharmaceutically acceptable salt thereof, and can be lyophilized. The unit dosage of phosphorylated dimethylaminoarglabin or a pharmaceutically acceptable salt thereof can range from about 0.5 mg/kg to about 7 mg/kg, and is particularly useful from about 3.4 mg/kg to about 4.0 mg/kg.
The invention also features an article of manufacture including packaging material and a pharmaceutical agent contained within the packaging material. The packaging material includes a label that indicates that the pharmaceutical agent can be used for suppressing tumor growth in a human. The pharmaceutical agent includes a phosphosesquiterpene. Phosphorylated dimethylaminoarglabin or a pharmaceutically acceptable salt thereof are particularly useful phosphosesquiterpenes.
As used herein, phosphate or phospho group refers to pyrophosphate or orthophosphate. A phosphorylation product refers to a product obtained from a phosphorylation reaction performed on a sesquiterpene lactone. xe2x80x9cSkeletal structurexe2x80x9d refers to the carbon framework of a compound. For example, the 15-carbon frameworks found in formulas A-H are skeletal structures. As used herein, two carbon atoms linked to each other via an oxy linkage means that an oxirane or epoxide structure is formed. The term xe2x80x9cshort chain alkylxe2x80x9d includes methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl, and tert-butyl substituents. xe2x80x9cShort chain alkanoyloxyxe2x80x9d includes acetoxy, propioinyloxy, n-butyryloxy, iso-butyryloxy, sec-butyryloxy, and tert-butyryloxy substituents. xe2x80x9cShort chain alkanoic acidxe2x80x9d includes formic acid, acetic acid, propionic acid, n-butyric, iso-butyric, sec-butyric, and tert-butyric acid substituents. xe2x80x9cOptionally substituted alklyamino saltsxe2x80x9d include N,N-di(short chain alkyl)amino hydrogen chloride salts.
xe2x80x9cM+xe2x80x9d refers to any chemical entity or combination of chemical entities that is able to neutralize any negative charges that are present in the phosphosesquiterpenes of the invention. These negative charges typically arise from dissociation of acidic hydrogens from heteroatoms present on the compounds, for example, the oxygen atoms of carboxylic or phosphate substituents. It is recognized that these acidic hydrogens may be fully ionized, partially ionized, or un-ionized under different conditions. Thus M+ can be any combination of hydrogen, or any alkali metal cation, preferably sodium or potassium. The chemical entities represent by M+ may also have the ability to neutralize multiple negative charges in the phosphosesquiterpenes of the invention.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
Other features and advantages of the invention will be apparent from the following detailed description, and from the claims.